Remodeling Isoprene Pyrophosphate Metabolism for Promoting Terpenoids Bioproduction

Terpenoids are the largest family of natural products.They are made from the building block isoprene pyrophosphate(IPP),and their bioproduction using engineered cell factories has received a great deal of attention.To date,the insufficient metabolic supply of IPP remains a great challenge for the efficient synthesis of terpenoids.In this work,we discover that the imbalanced metabolic flux distribution between the central metabolism and the IPP supply hinders IPP accumulation in Bacillus subtilis(B.subtilis).Therefore,we remodel the IPP metabolism using a series of genetically encoded two-input-multioutput(TIMO)circuits that are responsive to pyruvate or/and malonyl-CoA,resulting in an IPP pool that is significantly increased by up to four-fold.As a proof-of-concept validation,we design an IPP metabolism remodeling strategy to improve the production of three valuable terpenoids,including menaquinone-7(MK-7,4.1-fold),lycopene(9-fold),andβ-carotene(0.9-fold).In particular,the titer of MK-7 in a 50-L bioreactor reached 1549.6 mg·L^(-1),representing the highest titer reported so far.Thus,we propose a TIMO genetic circuits-assisted IPP metabolism remodeling framework that can be generally used for the synergistic fine-tuning of complicated metabolic modules to achieve the efficient bioproduction of terpenoids.

Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Alpha-lactalbumin(α-LA)is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants.In this study,Bacillus subtilis RIK1285 harboring AprE signal peptide(SP)was selected as the original strain for the production ofα-LA.It was found thatα-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant.The original strain most likely only producedα-LA intracellular,but not extracellular.To improve the expression and secretion ofα-LA in RIK1285,a library of 173 homologous SPs from the B.subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285.SP YjcN was determined to be the best signal peptide.Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide.In addition,different promoters(P_(aprE),P_(43),and P_(glv))were compared and applied.The results indicated that the strain RIK1285-pBE-P_(glv)-YjcN-LALBA had the highestα-LA yield,reaching 122.04μg/mL.This study demonstrates successful expression and secretion of humanα-LA in B.subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

Minimizing endogenous cryptic plasmids to construct antibiotic-free expression systems for Escherichia coli Nissle 1917

The probiotic bacterium Escherichia coli Nissle 1917(EcN)holds significant promise for use in clinical and biological industries.However,the reliance on antibiotics to maintain plasmid-borne genes has overshadowed its benefits.In this study,we addressed this issue by engineering the endogenous cryptic plasmids pMUT1 and pMUT2.The non-essential elements were removed to create more stable derivatives pMUT1NR△and pMUT2HBC△.Synthetic promoters by integrating binding motifs on sigma factors were further constructed and applied for expression of Bacteroides thetaiotaomicron heparinaseⅢand the biosynthesis of ectoine.Compared to traditional antibiotic-dependent expression systems,our newly constructed antibiotic-free expression systems offer considerable advantages for clinical and synthetic biology applications.

Food synthetic biology-driven protein supply transition: From animal-derived production to microbial fermentation

Animal-derived protein production is one of the major traditional protein supply methods,which continues to face increasing challenges to satisfy global needs due to population growth,augmented individual protein consumption,and aggravated environmental pollution.Thus,ensuring a sustainable protein source is a considerable challenge.The emergence and development of food synthetic biology has enabled the establishment of cell factories that effectively synthesize proteins,which is an important way to solve the protein supply problem.This review aims to discuss the existing problems of traditional protein supply and to elucidate the feasibility of synthetic biology in the process of protein synthesis.Moreover,using artificial bioengineered milk and artificial bioengineered eggs as examples,the progress of food protein supply transition based on synthetic biology has been systematically summarized.Additionally,the future of food synthetic biology as a potential source of protein has been also discussed.By strengthening and innovating the application of food synthetic biology technologies,including genetic engineering and high-throughput screening methods,the current limitations of artificial foods for protein synthesis and production should be addressed.Therefore,the development and industrial production of new food resources should be explored to ensure safe,high-quality,and sustainable global protein supply.

Current progress and prospects of enzyme technologies in future foods

Enzyme technologies are widely used in the food industry due to their advantages of high efficiency,specificity,and safety.Recently,“future foods”is emerging as a new research hotspot with healthier foods that are more nutritious,delicious,and sustainable;however,these foods still have problems with texture,nutrition,and flavor.Advances in enzyme technology have enabled the development of new tools and approaches to better manipulate food textures and nutritional aspects.In this review,we summarize enzyme technology applications in future food production,focusing on food texture,safety,and flavors.Furthermore,we discuss the prospects of enzyme-based technologies for future food production,including the modification of enzyme activities,the development of suitable food-grade hosts for enzyme production,and the optimiza-tion synergistic multi-enzyme systems.

A review on current conventional and biotechnical approaches to enhance biosynthesis of steviol glycosides in Stevia rebaudiana

Stevia rebaudiana Bertoni is commonly called stevia and mostly found in the north east regions of South America.It is an herbaceous and shrubby plant belonging to the Asteraceae family.Stevia is considered as a natural sweetener and a commercially important plant worldwide.The leaves of S.rebaudiana contain steviol glycosides(SGs)which are highly potent and non-caloric sweeteners.The sweetening property of S.rebaudiana is contributed to the presence of these high potency,calorie free steviol glycosides.SGs are considerably suitable for replacing sucrose and other artificial sweetening agents which are used in different industries and pharmaceuticals.SGs amount in the plant mostly varies from 8%to 10%,and the enhancement of SGs is always in demand.These glycosides have the potential to become healthier alternatives to other table sugars for having desirable taste and zero calories.SGs are almost 300 times sweeter than sucrose.Being used as alternative sugar intensifier the commercial value of this plant in biopharmaceutical,food and beverages industries and in international market is increasing day by day.SGs have made stevia an important part of the medicinal world as well as the food and beverage industry,but the limited production of plant material is not fulfilling the higher global market demand.Therefore,researchers are working worldwide to increase the production of important SGs through the intercession of different biotechnological approaches in S.rebaudiana.This review aims to describe the emerging biotechnological strategies and approaches to understand,stimulate and enhance biosynthesis of secondary metabolites in stevia.Conventional and biotechnological methods for the production of steviol glycosides have been briefly reviewed and discussed.

Synergetic engineering of Escherichia coli for efficient production of L-tyrosine

L-Tyrosine,an aromatic non-essential amino acid,is the raw material for many important chemical products,including levodopa,resveratrol,and hydroxytyrosol.It is widely used in the food,drug,and chemical industries.There are many studies on the synthesis of L-tyrosine by microorganisms,however,the low titer of L-tyrosine limited the industrial large-scale production.In order to enhance L-tyrosine production in Escherichia coli,the expression of key enzymes in the shikimate pathway was up-or down-regulated.The L-tyrosine transport system and the acetic acid biosynthesis pathway were modified to further enhance L-tyrosine production.In addition,the phosphoketolase pathway was introduced in combination with cofactor engineering to redirect carbon flux to the shikimate pathway.Finally,after adaptive laboratory evolution to low pH an optimal strain was obtained.The strain can produce 92.5 g/L of L-tyrosine in a 5-L fermenter in 62 h,with a yield of 0.266 g/g glucose.

Yeast surface display of leech hyaluronidase for the industrial production of hyaluronic acid oligosaccharides

Leech hyaluronidase(LHyal)is a hyperactive hyaluronic acid(HA)hydrolase that belongs to the hyaluronoglu-curonidase family.Traditionally,LHyal is extracted from the heads of leeches,but the recent development of the Pichia pastoris recombinant LHyal expression method permitted the industrial production of size-specific HA oligosaccharides.However,at present LHyal expressed by recombinant yeast strains requires laborious protein purification steps.Moreover,the enzyme is deactivated and removed after single use.To solve this problem,we developed a recyclable LHyal biocatalyst using a yeast surface display(YSD)system.After screening and charac-terization,we found that the cell wall protein Sed1p displayed stronger anchoring to the P.pastoris cell wall than other cell wall proteins.By optimizing the type and length of the linkers between LHyal and Sed1p,we increased the activity of enzymes displayed on the P.pastoris cell wall by 50.34%in flask cultures.LHyal-(GGGS)6-Sed1p activity further increased to 3.58×105 U mL−1 in fed-batch cultivation in a 5 L bioreactor.Enzymatic prop-erty analysis results revealed that the displayed LHyal-(GGGS)6-Sed1p generated the same oligosaccharides but exhibited higher thermal stability than free LHyal enzyme.Moreover,displayed LHyal-(GGGS)6-Sed1p could be recovered easily from HA hydrolysis solutions via low-speed centrifugation and could be reused at least 5 times.YSD of LHyal not only increased the utilization efficiency of the enzyme but also simplified the purification pro-cess for HA oligosaccharides.Thus,this study provides an alternative approach for the industrial preparation of LHyal and HA oligosaccharides.

Microbial chassis design and engineering for production of amino acids used in food industry

Rational microbial chassis design and engineering for improving production of amino acids have attracted a considerable attention.l-glutamate,l-lysine,l-threonine and l-tryptophan are the main amino acids demanded in the food industry.Systems metabolic engineering and synthetic biology engineering generally are believed as the comprehensive engineering approaches to obtain rationally designed strains and construct high-performance platforms for amino acids.The strate-gies focus on microbial chassis characterization optimization,precise metabolic engineering such as promoter engineer-ing,modular pathway engineering,transporter engineering,and dynamic switch systems application,and global genome streamline engineering to reduce cell burden.In this review,we summarized the efficient engineering strategies to optimize Corynebacterium glutamicum and Escherichia coli cell factories for improving the production of l-glutamate,l-lysine,l-threonine,and l-tryptophan.

Engineering Saccharomyces cerevisiae for enhanced(–)-α-bisabolol production

(–)-α-Bisabolol is naturally occurring in many plants and has great potential in health products and pharma-ceuticals.However,the current extraction method from natural plants is unsustainable and cannot fulfil the increasing requirement.This study aimed to develop a sustainable strategy to enhance the biosynthesis of(–)-α-bisabolol by metabolic engineering.By introducing the heterologous gene MrBBS and weakening the competitive pathway gene ERG9,a de novo(–)-α-bisabolol biosynthesis strain was constructed that could produce 221.96 mg/L(–)-α-bisabolol.Two key genes for(–)-α-bisabolol biosynthesis,ERG20 and MrBBS,were fused by a flexible linker(GGGS)3 under the GAL7 promoter control,and the titer was increased by 2.9-fold.Optimization of the mevalonic acid pathway and multi-copy integration further increased(–)-α-bisabolol production.To promote product efflux,overexpression of PDR15 led to an increase in extracellular production.Combined with the optimal strategy,(–)-α-bisabolol production in a 5 L bioreactor reached 7.02 g/L,which is the highest titer reported in yeast to date.This work provides a reference for the efficient production of(–)-α-bisabolol in yeast.

Engineering Escherichia coli for high-yield production of ectoine

Ectoine is a natural macromolecule protector and synthesized by some extremophiles.It provides protections against radiation-mediated oxidative damages and is widely used as a bioactive ingredient in pharmaceutics and cosmetics.To meet its growing commercial demands,we engineered Escherichia coli strains for the high-yield production of ectoine.The ectABC gene cluster from the native ectoine producer Halomonas elongata was intro-duced into different Escherichia coli(E.Coil)strains via plasmids and 0.8 g L^(-1)of ectoine was produced in flask cultures by engineered E.coli BL21(DE3).Subsequently,we designed the ribosome-binding sites of the gene cluster to fine-tune the expressions of genes ectA,ectB,and ectC,which increased the ectoine yield to 1.6 g L^(-1).After further combinatorial overexpression of Corynebacterium glutamicum aspartate kinase mutant(G1A,C932T)and the H.elongate aspartate-semialdehyde dehydrogenase to increase the supply of the precursor,the titer of ectoine reached to 5.5 g L^(-1)in flask cultures.Finally,the engineered strain produced 60.7 g L^(-1)ectoine in fed-batch cultures with a conversion rate of 0.25 g/g glucose.

Characterization of putative mannoprotein in Kluyveromyces lactis for lactase production

Lactase is a member of theβ-galactosidase family of enzymes that can hydrolyze lactose into galactose and glucose.However,extracellular lactase production was still restricted to the process of cell lysis.In this study,lactase-producing Kluyveromyces lactis JNXR-2101 was obtained using a rapid and sensitive method based on the fluorescent substrate 4-methylumbelliferyl-β-D-galactopyranoside.The purified enzyme was identified as a neutral lactase with an optimum pH of 9.To facilitate extracellular production of lactase,a putative mannoprotein KLLA0_E01057g of K.lactis was knocked out.It could effectively promote cell wall degradation and lactase production after lyticase treatment,which showed potential on other extracellular enzyme preparation.After optimizing the fermentation conditions,the lactase yield from mannoprotein-deficient K.lactis JNXR-2101ΔE01057g reached 159.62 U/mL in a 5-L fed-batch bioreactor.

Design of artificial small regulatory trans-RNA for gene knockdown in Bacillus subtilis

Bacillus subtilis as the Gram-positive model bacterium has been widely used in synthetic biology and biotechnology while the regulatory RNA tools for B.subtilis are still not fully explored.Here,a bottom-up approach is proposed for designing artificial trans-acting sRNAs.By engineering the intrinsic sRNA SR6,a minimized core scaffold structure consisting of an 8 bp stem,a 4 nt loop,and a 9 nt polyU tail was generated and proven to be sufficient for constructing sRNAs with strong repression activity(83%).Moreover,we demonstrate this artificial sRNA system functions well in an hfq-independent manner and also achieves strong repression efficiency in Escherichia coli(above 80%).A structure-based sRNA design principle was further developed for the automatic generation of custom sRNAs with this core scaffold but various sequences,which facilitates the manipulation and avoids structure disruption when fusing any base-pairing sequence.By applying these auto-designed sRNAs,we rapidly modified the cell morphology and biofilm formation,and regulated metabolic flux toward acetoin biosynthesis.This sRNA system with cross-species regulatory activities not only enriched the gene regulation toolkit in synthetic biology for B.subtilis and E.coli but also enhanced our understanding of trans-acting sRNAs.

Crude enzyme immobilization‑based cell‑free system for efficient N‑acetylneuraminic acid biosynthesis aided by N‑terminal coding sequence screening

N-acetylneuraminic acid(NeuAc)is an important nutrient that plays a key role in brain development in infants NeuAc is mainly produced by extraction from natural resources such as edible birds’s nests,crucian eggs,caviar and human breast milk.The extraction process is complicated,resulting in the disadvantages of low NeuAc content and low recovery rate.In this study,a crude enzyme immobilization-based cell-free system(CEICFS)was developed for efficient NeuAc biosynthesis.First,N-terminal coding sequences that improved the expression levels of N-acetylglucosamine-2-epimerase(AGE)and N-acetylneuraminic acid aldolase(NanA)were obtained by high-throughput screening.And these sequences resulted in up to 1.5-fold(1.2-fold)increase in AGE(NanA)enzyme levels.And then,a CEICFS for NeuAc biosynthesis was proposed by directly immobilizing crude enzyme containing AGE and NanA on amino resin.Subsequently,NeuAc production from GlcNAc using CEICFS in one reactor was carried out,resulting 68 g/L of NeuAc and the highest productivity of 6.8 g/L/h.Further,the enzyme activity was still higher than 75%after five repeated uses.The functional properties of CEICFS were studied and compared to those of the free enzyme,immobilization can extend the application of enzyme to some harsh environments,such as low temperature and acidic environment.Therefore,CEICFS with excellent heat resistance,storage stability and reusability exhibit great potential for industrial application.

Regulatory RNAs in Bacillus subtilis:A review on regulatory mechanism and applications in synthetic biology

Bacteria exhibit a rich repertoire of RNA molecules that intricately regulate gene expression at multiple hierarchical levels,including small RNAs(sRNAs),riboswitches,and antisense RNAs.Notably,the majority of these regulatory RNAs lack or have limited protein-coding capacity but play pivotal roles in orchestrating gene expression by modulating transcription,post-transcription or translation processes.Leveraging and redesigning these regulatory RNA elements have emerged as pivotal strategies in the domains of metabolic engineering and synthetic biology.While previous investigations predominantly focused on delineating the roles of regulatory RNA in Gram-negative bacterial models such as Escherichia coli and Salmonella enterica,this review aims to summarize the mechanisms and functionalities of endogenous regulatory RNAs inherent to typical Gram-positive bacteria,notably Bacillus subtilis.Furthermore,we explore the engineering and practical applications of these regulatory RNA elements in the arena of synthetic biology,employing B.subtilis as a foundational chassis.

CRISPR/Cpf1–FOKI-induced gene editing in Gluconobacter oxydans

Gluconobacter oxydans is an important Gram-negative industrial microorganism that produces vitamin C and other products due to its efficient membrane-bound dehydrogenase system.Its incomplete oxidation system has many crucial industrial applications.However,it also leads to slow growth and low biomass,requiring further metabolic modification for balancing the cell growth and incomplete oxidation process.As a non-model strain,G.oxydans lacks efficient genome editing tools and cannot perform rapid multi-gene editing and complex metabolic network regulation.In the last 15 years,our laboratory attempted to deploy multiple CRISPR/Cas systems in different G.oxydans strains and found none of them as functional.In this study,Cpf1-based or dCpf1-based CRISPRi was constructed to explore the targeted binding ability of Cpf1,while Cpf1–FokI was deployed to study its nuclease activity.A study on Cpf1 found that the CRISPR/Cpf1 system could locate the target genes in G.oxydans but lacked the nuclease cleavage activity.Therefore,the CRISPR/Cpf1–FokI system based on FokI nuclease was constructed.Single-gene knockout with efficiency up to 100%and double-gene iterative editing were achieved in G.oxydans.Using this system,AcrVA6,the anti-CRISPR protein of G.oxydans was discovered for the first time,and efficient genome editing was realized.

Expression and antimicrobial activity of the recombinant bovine lactoferricin in Pichia pastoris

Lactoferricin,a multifunctional peptide located in the N-terminal region of lactoferrin,has a broad-spectrum bacteriostatic activity.It is a promising candidate as a food additive and immune fortification agent and does not have the risks associated with drug residues and drug resistance.First,we performed promoter and host cell screening to achieve the recombinant expression of lactoferricin in Pichia pastoris,showing an initial titer of 19.5 mg/L in P.pastoris X-33 using PAOX1 promoter.Second,we constructed a 0030-α hybrid signal peptide by fusing the 0030 signal peptide with the pro-sequence of α-factor secretory signal peptide.This further increased the production of lactoferricin,with a titer of 28.8 mg/L in the fermentation supernatant in the shaking flask.Next,we increased the expression of lactoferricin by fusing it with anionic antioxidant peptides.The neutralization of positive charges yielded a titer of 55.3 mg/L in the shaking flask,and a highest titer of 193.9 mg/L in a 3-L bioreactor.The antimicrobial activity analysis showed that recombinant-expressed lactoferricin exhibited potent antibacterial activity against Escherichia coli,Bacillus subtilis,and Staphylococcus aureus.This study provides a reference for the construction of microbial cell factories capable of efficiently synthesizing antimicrobial peptides.

Facilitating stable gene integration expression and copy number amplification in Bacillus subtilis through a reversible homologous recombination switch

Strengthening the expression level of integrated genes on the genome is crucial for consistently expressing key enzymes in microbial cell factories for efficient bioproduction in synthetic biology.In comparison to plasmid-based multi-copy expression,the utilization of chromosomal multi-copy genes offers increased stability of expression level,diminishes the metabolic burden on host cells,and enhances overall genetic stability.In this study,we developed the“BacAmp”,a stabilized gene integration expression and copy number amplification system for high-level expression in Bacillus subtilis,which was achieved by employing a combination of repressor and non-natural amino acids(ncAA)-dependent expression system to create a reversible switch to control the key gene recA for homologous recombination.When the reversible switch is turned on,genome editing and gene amplification can be achieved.Subsequently,the reversible switch was turned off therefore stabilizing the gene copy number.The stabilized gene amplification system marked by green fluorescent protein,achieved a 3-fold increase in gene expression by gene amplification and maintained the average gene copy number at 10 after 110 generations.When we implemented the gene amplification system for the regulation of N-acetylneuraminic acid(NeuAc)synthesis,the copy number of the critical gene increased to an average of 7.7,which yielded a 1.3-fold NeuAc titer.Our research provides a new avenue for gene expression in synthetic biology and can be applied in metabolic engineering in B.subtilis.

Efficient biosynthesis of creatine by whole-cell catalysis from guanidinoacetic acid in Corynebacterium glutamicum

Creatine is a naturally occurring derivative of an amino acid commonly utilized in functional foods and pharmaceuticals.Nevertheless,the current industrial synthesis of creatine relies on chemical processes,which may hinder its utilization in certain applications.Therefore,a biological approach was devised that employs whole-cell biocatalysis in the bacterium Corynebacterium glutamicum,which is considered safe for use in food production,to produce safe-for-consumption creatine.The objective of this study was to identify a guanidinoacetate N-methyltransferase(GAMT)with superior catalytic activity for creatine production.Through employing whole-cell biocatalysis,a gamt gene from Mus caroli(Mcgamt)was cloned and expressed in C.glutamicum ATCC 13032,resulting in a creatine titer of 3.37 g/L.Additionally,the study employed a promoter screening strategy that utilized nine native strong promoters in C.glutamicum to enhance the expression level of GAMT.The highest titer was achieved using the P1676 promoter,reaching 4.14 g/L.The conditions of whole-cell biocatalysis were further optimized,resulting in a creatine titer of 5.42 g/L.This is the first report of successful secretory creatine expression in C.glutamicum,which provides a safer and eco-friendly approach for the industrial production of creatine.

A CRISPR/Cas9-based visual toolkit enabling multiplex integration at specific genomic loci in Aspergillus niger

Aspergillus niger is a highly versatile fungal strain utilized in industrial production.The expression levels of recombinant genes in A.niger can be enhanced by increasing the copy number.Nevertheless,given the prolonged gene editing cycle of A.niger,a“one-step”strategy facilitating the simultaneous integration of recombinant genes into multiple genomic loci would provide a definitive advantage.In our previous study,a visual multigene editing system(VMS)was designed to knock out five genes,employing a tRNA-sgRNA array that includes the pigment gene albA and the target genes.Building upon this system,hybrid donor DNAs(dDNAs)were introduced to establish a clustered regularly interspaced short palindromic repeats(CRISPR)-based multiplex integration toolkit.Firstly,a CRISPR-Cas9 homology-directed repair(CRISPR-HDR)system was constructed in A.niger by co-transforming the CRISPR-Cas9 plasmid(with a highly efficient sgRNA)and the dDNA,resulting in precise integration of recombinant xylanase gene xynA into the target loci(theβ-glucosidase gene bgl,the amylase gene amyA,and the acid amylase gene ammA).Subsequently,the length of homology arms in the dDNA was optimized to achieve 100%editing efficiency at each of the three gene loci.To achieve efficient multiplex integration in A.niger,the CRISPR plasmid pLM2 carrying a sgRNA-tRNA array was employed for concurrent double-strand breaks at multiple loci(bgl,amyA,ammA,and albA).Hybrid dDNAs were then employed for repair,including dDNA1-3(containing xynA expression cassettes without selection markers)and dDNAalbA(for albA knockout).Among the obtained white colonies(RLM2′),23.5%exhibited concurrent replacement of the bgl,amyA,and ammA genes with xynA(three copies).Notably,the xynA activity obtained by simultaneous insertion into three loci was 48.6%higher compared to that obtained by insertion into only the bgl locus.Furthermore,this multiple integration toolkit successfully enhanced the expression of endogenous pectinase pelA and Candida antarctica lipase CALB.Hence,the combined application of VMS and the CRISPR-HDR system enabled the simultaneous application of multiple selection markers,facilitating the rapid generation in the A.niger cell factories.